Margriet J. Vervoordeldonk

Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice

OBJECTIVE The parasympathetic nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor alpha (TNFalpha), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis. METHODS To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen-induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)-injected nicotine and the specific alpha7nAChR agonist AR-R17779. RESULTS Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFalpha expression in synovial tissue. Both IP-injected nicotine and AR-R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFalpha levels in both plasma and synovial tissue. The effect of AR-R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction. CONCLUSION These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.

Depletion of Alveolar Macrophages Exerts Protective Effects in Pulmonary Tuberculosis in Mice

Mycobacterium tuberculosis bacilli are intracellular organisms that reside in phagosomes of alveolar macrophages (AMs). To determine the in vivo role of AM depletion in host defense against M. tuberculosis infection, mice with pulmonary tuberculosis induced by intranasal administration of virulent M. tuberculosis were treated intranasally with either liposome-encapsulated dichloromethylene diphosphonate (AM− mice), liposomes, or saline (AM+ mice). AM− mice were completely protected against lethality, which was associated with a reduced outgrowth of mycobacteria in lungs and liver, and a polarized production of type 1 cytokines in lung tissue, and by splenocytes stimulated ex vivo. AM− mice displayed deficient granuloma formation, but were more capable of attraction and activation of T cells into the lung and had increased numbers of pulmonary polymorphonuclear cells. These data demonstrate that depletion of AMs is protective during pulmonary tuberculosis.

Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.

Evaluation of therapeutic targets in animal models of arthritis: How does it relate to rheumatoid arthritis?

Introduction Rheumatoid arthritis (RA) is a chronic, systemic, immune-mediated inflammatory disease associated with decreased life expectancy and quality of life. RA is characterized by chronic inflammation and synovial hyperplasia leading to destruction of cartilage and bone. A better understanding of the pathophysiology of RA has led to significant improvements in its treatment. However, many questions remain with respect to the pathogenesis of RA, and disease remission is achieved in only a minority of patients. Therefore, there is still a need to develop novel antirheumatic therapies. Animal models of RA complement descriptive studies of well-defined patient samples and in vitro studies and represent important tools for the development and evaluation of new treatment options. This review article addresses the immunologic characteristics of and similarities and differences between a variety of animal models of human RA. Commonly used animal models such as collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA), and a model of spontaneous arthritis using tumor necrosis factor (TNF)–transgenic mice are described. We also discuss the less frequently used models of streptococcal cell wall (SCW)–induced arthritis, proteoglycan-induced arthritis (PGIA), and K/BxN-transgenic mice, including the serum transfer–induced (STIA) model, to indicate the variety of animal models with specific characteristics that are available and that could be used to study specific components and stages of the inflammation process. Furthermore, we describe the effects of some targeted interventions, comparing animal studies with clinical trials. Finally, the importance of choosing a suitable animal model of arthritis for screening the preclinical efficacy of newly developed therapeutics is addressed.

Animal models for arthritis: innovative tools for prevention and treatment

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive ‘pathogenesis map’ that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in ‘omics’ profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

Restoring the Balance of the Autonomic Nervous System as an Innovative Approach to the Treatment of Rheumatoid Arthritis

The immunomodulatory effect of the autonomic nervous system has raised considerable interest over the last decades. Studying the influence on the immune system and the role in inflammation of the sympathetic as well as the parasympathetic nervous system not only will increase our understanding of the mechanism of disease, but also could lead to the identification of potential new therapeutic targets for chronic immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA). An imbalanced autonomic nervous system, with a reduced parasympathetic and increased sympathetic tone, has been a consistent finding in RA patients. Studies in animal models of arthritis have shown that influencing the sympathetic (via α- and β-adrenergic receptors) and the parasympathetic (via the nicotinic acetylcholine receptor α7nAChR or by electrically stimulating the vagus nerve) nervous system can have a beneficial effect on inflammation markers and arthritis. The immunosuppressive effect of the parasympathetic nervous system appears less ambiguous than the immunomodulatory effect of the sympathetic nervous system, where activation can lead to increased or decreased inflammation depending on timing, doses and kind of adrenergic agent used. In this review we will discuss the current knowledge of the role of both the sympathetic (SNS) and parasympathetic nervous system (PNS) in inflammation with a special focus on the role in RA. In addition, potential antirheumatic strategies that could be developed by targeting these autonomic pathways are discussed.

Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor α7 subunit gene knockout mice

Background The α7 subunit of nicotinic acetylcholine receptors (α7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the α7nAChR can reduce the severity of arthritis in murine collagen-induced arthritis (CIA). Objective To provide more insight into the role of the α7nAChR in the pathogenesis of arthritis by investigating the effect of the absence of α7nAChR in CIA in α7-deficient (α7nAChR-/-) compared with wild-type (WT) mice. Methods CIA was induced in α7nAChR-/- and WT littermate mice at day 0 by immunisation with chicken collagen type II (cCII) followed by a booster injection with cCII on day 20. Mice were killed on day 44 or day 63 and arthritis activity as well as radiological and histological damage were scored. The effects on the immune response were evaluated by measurement of antigen-specific antibodies and cytokines, and evaluation of the effects on antigen-specific stimulated spleen cells. Results In α7nAChR-/- mice a significant increase in the incidence and severity of arthritis as well as increased synovial inflammation and joint destruction were seen. Exacerbation of CIA was associated with elevated systemic proinflammatory cytokines and enhanced T-helper cell 1 (Th1)-cytokine and tumour necrosis factor α production by spleen cells. Moreover, a specific decrease in the collagen-specific ‘Th1-associated’ IgG2a response was seen, whereas IgG1 titres were unaffected. Conclusions The results presented here indicate that immune cell function in a model of rheumatoid arthritis is regulated by the cholinergic system and, at least in part, mediated by the α7nAChR.

Selective inhibition of NF‐κB in dendritic cells by the NEMO‐binding domain peptide blocks maturation and prevents T cell proliferation and polarization

Dendritic cells (DC) are the only antigen‐presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF‐κB dependent, we hypothesized that blocking NF‐κB activity in DC by selectively targeting the inhibitor of κB (IκB) kinase (IKK) complex using the novel NF‐κB inhibitor NEMO‐binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte‐derived DC to test the biological effects of the NBD peptide in vitro. NF‐κB inhibition by the NBD peptide resulted in blockade of IKK‐mediated IκBα phosphorylation and subsequent nuclear translocation and DNA binding of NF‐κB p65 in DC. In addition, IL‐6, IL‐12, and TNF‐α production was dose‐dependently blocked and NBD peptide treatment also led to a strong reduction of LPS‐induced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF‐κB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses.

Neurostimulation of the Cholinergic Anti-Inflammatory Pathway Ameliorates Disease in Rat Collagen-Induced Arthritis

Introduction The inflammatory reflex is a physiological mechanism through which the nervous system maintains immunologic homeostasis by modulating innate and adaptive immunity. We postulated that the reflex might be harnessed therapeutically to reduce pathological levels of inflammation in rheumatoid arthritis by activating its prototypical efferent arm, termed the cholinergic anti-inflammatory pathway. To explore this, we determined whether electrical neurostimulation of the cholinergic anti-inflammatory pathway reduced disease severity in the collagen-induced arthritis model. Methods Rats implanted with vagus nerve cuff electrodes had collagen-induced arthritis induced and were followed for 15 days. Animals underwent active or sham electrical stimulation once daily from day 9 through the conclusion of the study. Joint swelling, histology, and levels of cytokines and bone metabolism mediators were assessed. Results Compared with sham treatment, active neurostimulation of the cholinergic anti-inflammatory pathway resulted in a 52% reduction in ankle diameter (p = 0.02), a 57% reduction in ankle diameter (area under curve; p = 0.02) and 46% reduction overall histological arthritis score (p = 0.01) with significant improvements in inflammation, pannus formation, cartilage destruction, and bone erosion (p = 0.02), accompanied by numerical reductions in systemic cytokine levels, not reaching statistical significance. Bone erosion improvement was associated with a decrease in serum levels of receptor activator of NF-κB ligand (RANKL) from 132±13 to 6±2 pg/mL (mean±SEM, p = 0.01). Conclusions The severity of collagen-induced arthritis is reduced by neurostimulation of the cholinergic anti-inflammatory pathway delivered using an implanted electrical vagus nerve stimulation cuff electrode, and supports the rationale for testing this approach in human inflammatory disorders.

Mice Lacking the Multidrug Resistance Protein 1 Are Resistant to Streptococcus pneumoniae-Induced Pneumonia

Leukotrienes (LTs) are considered important for antibacterial defense in the lung. Multidrug resistance protein 1 (mrp1) is a transmembrane protein responsible for the cellular extrusion of LTC4. To determine the role of mrp1 in host defense against pneumonia, mrp1−/− and wild-type mice were intranasally inoculated with Streptococcus pneumoniae. mrp1−/− mice displayed a diminished outgrowth of pneumococci in lungs and a strongly reduced mortality. These findings were related to an effect of mrp1 on LT metabolism, because survival was similar in mrp1−/− and wild-type mice treated with the 5-lipoxygenase-activating protein inhibitor MK-886. Although LTC4 levels remained low in the bronchoalveolar lavage fluid of mrp1−/− mice, LTB4 concentrations were higher than in wild-type mice. These elevated LTB4 concentrations were important for the relative protection of mrp1−/− mice, because the LTB4 antagonist LTB4-dimethyl amide abolished their survival advantage. In vitro experiments suggested that the intracellullar accumulation of LTC4 in mrp1−/− mice results in product inhibition of LTC4-synthase, diminishing substrate competition between LTA4-hydrolase (which yields LTB4) and LTC4-synthase for the available LTA4. We conclude that mrp1−/− mice are resistant against pneumococcal pneumonia by a mechanism that involves increased release of LTB4. These results identify mrp1 as a novel target for adjunctive therapy in pneumonia.